EP3980785A1 - Essai biologique d'évaluation d'une insuffisance cardiaque - Google Patents

Essai biologique d'évaluation d'une insuffisance cardiaque

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Publication number
EP3980785A1
EP3980785A1 EP20731073.1A EP20731073A EP3980785A1 EP 3980785 A1 EP3980785 A1 EP 3980785A1 EP 20731073 A EP20731073 A EP 20731073A EP 3980785 A1 EP3980785 A1 EP 3980785A1
Authority
EP
European Patent Office
Prior art keywords
monoclonal antibody
patient
seq
amino acid
collagen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20731073.1A
Other languages
German (de)
English (en)
Inventor
Federica GENOVESE
Morten Karsdal
Lei Zhao
David Gordon
Zhaoqing Wang
Julio Alonso Chirinos MEDINA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nordic Bioscience AS
Bristol Myers Squibb Co
University of Pennsylvania Penn
Original Assignee
Nordic Bioscience AS
Bristol Myers Squibb Co
University of Pennsylvania Penn
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Filing date
Publication date
Application filed by Nordic Bioscience AS, Bristol Myers Squibb Co, University of Pennsylvania Penn filed Critical Nordic Bioscience AS
Publication of EP3980785A1 publication Critical patent/EP3980785A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to immunoassays for detecting and/or monitoring a cardiovascular disease in a patient and/or assessing the likelihood of or the severity of a cardiovascular disease in a patient.
  • the cardiovascular disease may in particular be heart failure, and especially heart failure with preserved ejection fraction.
  • the immunoassay may be for assessing the likelihood of adverse outcomes of the cardiovascular disease.
  • the patient may be a patient undergoing therapy for the cardiovascular disease, such as a patient undergoing treatment with an aldosterone antagonist.
  • the present invention also relates to immunoassays for identifying patients suitable for treatment with an aldosterone antagonist.
  • HF heart failure
  • HFpEF preserved ejection fraction
  • the heterogeneity of the HFpEF syndrome has been identified as an important barrier to demonstrating the effectiveness of candidate pharmacologic interventions.
  • HFpEF heterogenous nature of HFpEF
  • different degrees of contribution from various pathophysiological processes may unfavorably influence average responses to pharmacologic therapies tested in clinical trials. Therefore, the availability of simple, non-invasive biomarkers capable of readily identifying relevant underlying specific biologic processes that can be targeted with pharmacologic interventions represents a promising approach to enhance our clinical and therapeutic approach to HFpEF 4 .
  • HFpEF hetererogeneity
  • Myocardial fibrosis is thought to play a role in the pathophysiology of HFpEF 5,6 . Increased fibrosis results from an excess of formation relative to degradation of collagen, ultimately leading increased interstitial collagen deposition in the interstitium. Increased myocardial extracellular matrix deposition has been demonstrated in HFpEF in autopsy specimens and in vivo studies 6'8 and has been shown to correlate with LV passive stiffening and diastolic dysfunction in this condition 5,8 . Myocardial fibrosis may also contribute to reduced coronary flow reserve 6,9 , ventricular dyssynchrony and a propensity to arrhythmia 10,11 . Given the role of myocardial fibrosis in HFpEF, simple fibrotic biomarkers that reflect the underlying dynamic process of fibrosis progression or regression of fibrosis would be highly valuable 10 .
  • ECVF extracellular volume fraction
  • fibrosis is not just fibrosis
  • ECM remodeling of different compartments and collagen types may have different biologic and prognostic implications 15 .
  • differential associations of collagen neoepitope fragments and liver fibrosis has been reported in chronic hepatitis B vs. hepatitis C.
  • myocardial fibrosis is thought to be important in HFpEF, extracardiac fibrosis may also play an important role.
  • fibrofatty infiltration of skeletal muscle has been reported in HFpEF 16 .
  • fibrosis may also occur in the arterial wall, the kidney and the liver dysfunction, all of which may contribute to adverse outcomes in this population.
  • Type III collagen is expressed in most of the type I collagen containing tissues except for bone, and is an important component of connective tissues, muscle tissues and skin. Collagen type III is essential for collagen type I fibrillogenesis in the cardiovascular system and other organs. During fibrillar assembly the N-terminal propeptide of type III procollagen (which consists of three identical ochains with a total molecular weight of 42 kDa) is cleaved off by specific N- proteases prior to incorporation of the mature collagen in the extracellular matrix (ECM). The cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule.
  • ECM extracellular matrix
  • PIIINP is the N-terminal propeptide of collagen type III, which is removed during mature type III collagen synthesis.
  • the level of the N-terminal propeptide of type III collagen (PIIINP) in a suitable sample can be a marker of formation and/or degradation of collagen type III.
  • PRO-C3 is a biomarker for formation of collagen type III, comprising a C-terminal neo-epitope of the N-terminal propeptide of type III collagen (i.e. a C-terminal neo-epitope of PIIINP), which neo-epitope is formed after the cleavage of the pro-peptide from the pro-collagen by ADAMTS- 2.
  • the PRO-C3 biomarker, and a PRO-C3 assay are described in W02014/170312.
  • the assay utilizes a monoclonal antibody that specifically binds to the C- terminus 10 amino acid sequence of PIIINP, and so targets the free C-terminal end of the N- terminal pro-peptide that is formed after cleavage 19 .
  • PRO-C3 is a well explored biomarker of liver fibrosis, related both to fibrotic burden in the liver and to progression of fibrosis and adverse outcome in patients with different liver indications 20 26 .
  • Collagen Type VI is a unique extracellular collagen which can form an independent microfibrillar network in the basement membrane of cells. It can interact with other matrix proteins including collagens, biglycan, and proteoglycans.
  • type VI collagen is part of the sarcolemma and is involved in anchoring the muscle fiber into the intramuscular extracellular matrix, and so is involved in force transmission.
  • mutations in type VI collagen can cause Bethlem myopathy and Ullrich congenital muscular dystrophy. It has been reported that the C-terminal amino acid sequence of the type VI collagen a3 chain is cleaved off from the mature type VI microfibril after secretion. However, Type VI collagen is not just involved in muscles and muscle loss.
  • microflamentous interstitial type VI collagen a triple helical molecule composed of the constituent chains cd (VI), a2(VI), and a3(VI), is expressed in most connective tissues and prominently in adipose tissue, where it anchors cells through its interconnections with other ECM proteins.
  • the triple-helical core of type VI collagen is proteolytically released from the pro-peptide, and cleavage of the C-terminal pro-peptide of the a3(VI) chain generates endotrophin, an adipokine.
  • PRO-C6 is a biomarker for formation of collagen type VI and endotrophin release, comprising a C-terminal epitope of the C5 domain of the a3 chain of type VI collagen that is cleaved off when a novel collagen type VI molecule assembles in the extracellular matrix, and which C-terminal epitope is also a C-terminal epitope of the bioactive fragment endotrophin.
  • the PRO-C6 biomarker, and a PRO-C6 assay are described in WO2016/156526.
  • the assay utilizes a monoclonal antibody that specifically binds to the C- terminus 10 amino acid sequence of the C5 domain of the a3 chain of collagen type VI.
  • Endotrophin s role as a pro-fibrotic, pro-inflammatory and pro-tumorigenic molecule has been observed in preclinical models of breast cancer and liver fibrosis 27 31 .
  • PRO-C6 has been established as a prognostic biomarker for mortality and disease progression in chronic kidney disease and diabetic kidney disease patients 32 34 and as a predictive marker for response to glucose lowering therapy in diabetic patients 35 .
  • Collagen IV is a type of collagen found primarily in the basal lamina of vessels.
  • the vascular wall consists of two major types of extracellular matrix: the basement membrane and the interstitial matrix.
  • the basement membrane is composed of 2 independent polymeric networks: one made of type IV collagen, and made of laminins, in addition to proteoglycans 36,37 and various other glycoproteins.
  • the collagen IV network in the basement membrane is highly cross-linked and considered to maintain mechanical stability.
  • Basal membranes also harbor matrix metalloproteases (MMPs), a large family of broad-spectrum proteases that function in degradation and remodeling of the basement membrane.
  • MMPs matrix metalloproteases
  • C4M is a biomarker for MMP-mediated degradation of type IV collagen, comprising an N- terminal neo-epitope of a fragment of type IV collagen formed by cleavage of the a1 (IV) chain by MMP12.
  • the C4M biomarker, and a C4M assay (specifically, a C4M ELISA) have been described previously 38 .
  • the assay utilizes a monoclonal antibody that specifically binds to said N-terminal neo-epitope.
  • PRO-C6 and PRO-C3 as biomarkers for cardiovascular diseases.
  • the levels of PRO-C6 and PRO-C3 in the circulation of a cohort of patients with heart failure with preserved ejection fraction (HFpEF) were examined.
  • Baseline PRO-C3 and PRO-C6 levels were analyzed and the relationship between biomarker and outcomes were investigated, and both PRO-C3 and PRO-C6 were found to be effective diagnostic and prognostic biomarkers of heart failure.
  • C4M as a biomarker for patients with cardiovascular disease who may be responsive to treatment with an aldosterone antagonist.
  • the levels of C4M in patients with HFpEF were examined, and C4M was found to identify patients more likely to exhibit a favorable response to treatment with the aldosterone antagonist Spironolactone.
  • the present invention provides a method of immunoassay for detecting and/or monitoring a cardiovascular disease in a patient and/or assessing the likelihood of or the severity of a cardiovascular disease in a patient, wherein said method comprises:
  • step (ii) detecting and determining the amount of binding between each monoclonal antibody used in step (i) and peptides in the sample or samples, and (iii) correlating said amount of binding of each monoclonal antibody as determined in step (ii) with values associated with normal healthy subjects and/or values associated with known disease severity and/or values obtained from said patient at a previous time point and/or a predetermined cut-off value.
  • the immunoassay may be, but is not limited to, a competition assay or a sandwich assay.
  • the immunoassay may, for example, be a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the cardiovascular disease may in certain embodiments be heart failure.
  • the cardiovascular disease may be heart failure with a preserved ejection fraction (HFpEF).
  • HFpEF preserved ejection fraction
  • the method may in certain embodiments be a method for assessing the severity of a cardiovascular disease in a patient that comprises assessing the likelihood of patient mortality and/or hospitalization as a result of the cardiovascular disease and/or a composite of adverse cardiovascular events.
  • the patient may, for example, be a patient undergoing a therapy for the cardiovascular disease.
  • the patient biofluid sample may be, but is not limited to, blood, serum, plasma, urine or amniotic fluid.
  • the biofluid is serum or plasma.
  • the term“monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art.
  • whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light” and one "heavy” chain.
  • the N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region.
  • the variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition.
  • the constant region allows the antibody to recruit cells and molecules of the immune system.
  • Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity.
  • a monoclonal antibody comprising any constant region known in the art can be used.
  • Human constant light chains are classified as kappa and lambda light chains.
  • Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the IgG isotype has several subclasses, including, but not limited to IgGI, lgG2, lgG3, and lgG4.
  • the monoclonal antibody may preferably be of the IgG isotype, including any one of IgGI, lgG2, lgG3 or lgG4.
  • the CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al.
  • Antibodies can be generated from B cell clones as described in the examples.
  • the isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype, or human IgGI , lgG2, lgG3 or lgG4 subclasses.
  • the amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells, and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody.
  • primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined.
  • the light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence.
  • the full length heavy and light chains can be generated and sequenced.
  • the biofluid sample is contacted with a monoclonal antibody which specifically binds to a C-terminal epitope of the C5 domain of the a3 chain of type VI collagen.
  • a monoclonal antibody specifically binds to the C-terminus amino acid sequence KPGVISVMGT (SEQ ID No: 1 ) (also referred to herein as the“PRO-C6 sequence”, or simply“PRO-C6”).
  • said monoclonal antibody does not recognize or specifically bind to an elongated version of said C-terminus amino acid sequence which is KPGVISVMGTA (SEQ ID No: 2), or to a truncated version of said C-terminus amino acid sequence which is KPGVISVMG (SEQ ID No: 3).
  • the ratio of the affinity of said antibody for the C-terminus amino acid sequence KPGVISVMGT (SEQ ID No: 1) to the affinity of said antibody for the elongated C-terminus amino acid sequence KPGVISVMGTA (SEQ ID No: 2), and/or for the truncated C-terminus amino acid sequence KPGVISVMG (SEQ ID No: 3), is at least 10 to 1 , and more preferably is at least 50 to 1 , at least 100 to 1 , at least 500 to 1 , at least 1 ,000 to 1 , at least 10,000 to 1 , at least 100,000 to 1 , or at least 1 ,000,000 to 1.
  • C-terminus refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof.
  • the monoclonal antibody that specifically binds to the PRO-C6 sequence may preferably comprises one or more complementarity-determining regions (CDRs) selected from:
  • CDR-L1 RSSQRIVHSNGITFLE (SEQ ID No: 4)
  • RVSNRFS SEQ ID No: 5
  • CDR-L3 FQGSHVPLT (SEQ ID No: 6)
  • CDR-H2 AINPHNGATSYNQKFSG (SEQ ID No: 8)
  • the antibody comprises at least 2, 3, 4, 5 or 6 of the above listed CDR sequences.
  • the monoclonal antibody light chain variable region comprises the CDR sequences CDR-L1 : RSSQRIVHSNGITFLE (SEQ ID No: 4)
  • RVSNRFS SEQ ID No: 5
  • CDR-L3 FQGSHVPLT (SEQ ID No: 6).
  • the monoclonal antibody light chain comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) RSSQR ⁇ VHSNG ⁇ TFLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED Z.GZ. YYCFQGSHVPLT (SEQ ID No: 10).
  • the monoclonal antibody heavy chain variable region comprises the CDR sequences CDR-H1 : DFNMN (SEQ ID No: 7)
  • CDR-H2 AINPHNGATSYNQKFSG (SEQ ID No: 8) and
  • the monoclonal antibody heavy chain comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics)
  • the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity.
  • the similar or identical amino acids may be contiguous or non-contiguous.
  • the framework sequences may contain one or more amino acid substitutions, insertions and/or deletions.
  • Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid.
  • a skilled person would understand which amino acids share similar chemical properties.
  • the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gin; Group 3 His, Arg, Lys; Group 4 Met, Leu, lie, Val, Cys; Group 5 Phe Thy Trp.
  • a program such as the CLUSTAL program can be used to compare amino acid sequences.
  • This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences.
  • the monoclonal antibody that specifically binds to the PRO- C6 sequence may comprise the light chain variable region sequence:
  • the biofluid sample is contacted with a monoclonal antibody which specifically binds to a C-terminal neo epitope of the N-terminal propeptide of type III collagen.
  • said monoclonal antibody specifically binds to a C-terminus amino acid sequence CPTGPQNYSP (SEQ ID No: 14) (also referred to herein as the “PRO-C3 sequence”, or simply “PRO-C3”).
  • the monoclonal antibody does not recognize or specifically bind to an elongated version of said C- terminus amino acid sequence which is CPTGPQNYSPQ (SEQ ID No: 15), or to a truncated version of said C-terminus amino acid sequence which is CPTGPQNYS (SEQ ID No: 16).
  • the ratio of the affinity of said antibody for the C-terminus amino acid sequence CPTGPQNYSP (SEQ ID No: 14) to the affinity of said antibody for the elongated C-terminus amino acid sequence CPTGPQNYSPQ (SEQ ID No: 15), and/or for the truncated C-terminus amino acid sequence CPTGPQNYS (SEQ ID No: 16), is at least 10 to 1 , and more preferably is at least 50 to 1 , at least 100 to 1 , at least 500 to 1 , at least 1 ,000 to 1 , at least 10,000 to 1 , at least 100,000 to 1 , or at least 1 ,000,000 to 1.
  • the monoclonal antibody that specifically binds to the PRO-C3 sequence may preferably comprises one or more complementarity-determining regions (CDRs) selected from:
  • CDR-L3 FQGAHDPPA (SEQ ID No: 19)
  • CDR-H2 YMNPYNDVPKNNAKFRG (SEQ ID No: 21 )
  • the antibody comprises at least 2, 3, 4, 5 or 6 of the above listed CDR sequences.
  • the monoclonal antibody light chain variable region comprises the CDR sequences CDR-L1 : RSSQNIVYSNGDTYFE (SEQ ID No: 17)
  • CDR-L2 KVSQRFS (SEQ ID No: 18) and CDR-L3: FQGAHDPPA (SEQ ID No: 19).
  • the monoclonal antibody light chain comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) RSSQNWYSNGDTYFEWYLQKPGQSPKLLIYKVSQRFSGVPDRFSGSGSGTDFTLKISRVETE DL G V Y YCFQGAHDPPA (SEQ ID No: 23).
  • the monoclonal antibody heavy chain variable region comprises the CDR sequences CDR-H1 : GYTFINYVIH (SEQ ID No: 20)
  • the monoclonal antibody heavy chain comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics)
  • GYTF I N YVI H WLKQKAGQGPEWIGYMNPYNDVPKmAKFRGKARL TSDRSS TTA YMELNSL TS EDSAVYYCARGGFFGPLSY (SEQ ID No: 24).
  • the monoclonal antibody that specifically binds to the PRO- C3 sequence may comprise the light chain variable region sequence:
  • the amount of binding of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the a3 chain of collagen type VI, and/or the amount of binding of the monoclonal antibody specific for the C-terminal neo-epitope of the N-terminal propeptide of type III collagen (PIIINP), are correlated with values associated with normal healthy subjects and/or with values associated with known disease severity and/or with values obtained from the patient at a previous point in time.
  • values associated with normal healthy subjects and/or values associated with known disease severity means standardised quantities determined by the method described supra for subjects considered to be healthy, i.e. without a cardiovascular disease, and/or standardised quantities determined by the method described supra for subjects known to have a cardiovascular disease with a known severity.
  • the amount of binding of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the a3 chain of collagen type VI, and/or the amount of binding of the monoclonal antibody specific for the C- terminal neo-epitope of N-terminal propeptide of type III collagen (PIIINP), are compared with one or more predetermined cut-off values.
  • the“cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of cardiovascular disease in a patient, or of cardiovascular disease of a particular level of severity, in that a measured value of biomarker binding in a patient sample that is at or above the statistical cutoff value corresponds to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence or likelihood of cardiovascular disease or of a particular level of severity of the disease.
  • the predetermined cut-off value for the amount of binding of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the a3 chain of collagen type VI is preferably at least 1 1.0 ng/mL, more preferably at least 16.0 ng/ml_.
  • the predetermined cut-off value for amount of binding of the monoclonal antibody specific for the C-terminal neo-epitope of PIIINP is preferably at least 10.0 ng/mL, more preferably at least 14.0 ng/mL.
  • a measured amount of binding of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the a3 chain of collagen type VI of at least 1 1 ng/mL or greater, and in particular at least 16.0 ng/mL or greater, may be determinative of cardiovascular disease and/or increased risk of hospitalisation or mortality.
  • a statistical cut-off value of at least 1 1.0 ng/mL, and more preferably at least 16.0 ng/mL it is possible to utilise the method of the invention to give a prognosis of cardiovascular disease and/or increased risk of hospitalisation or mortality with a high level of confidence.
  • a measured amount of binding of the monoclonal antibody specific for C-terminal neo-epitope of PIIINP of at least 10 ng/mL or greater, and in particular at least 14.0 ng/mL or greater may be determinative of cardiovascular disease and/or increased risk of hospitalisation or mortality, and by having a statistical cut-off value of at least 10.0 ng/mL PRO-C3, and more preferably at least 14.0 ng/mL it is possible to utilise the method of the invention to give a prognosis of cardiovascular disease and/or increased risk of hospitalisation or mortality with a high level of confidence. Applying such statistical cut-off values are particularly advantageous as it results in a standalone diagnostic assay; i.e.
  • cardiovascular disease removes the need for any direct comparisons with healthy individuals and/or patients with known disease severity in order to arrive at a diagnostic conclusion.
  • This may also be particularly advantageous when utilising the assay to evaluate patients that already have medical signs or symptoms that are generally indicative of cardiovascular disease (e.g. as determined by a physical examination and/or consultation with a medical professional) as it may act as a quick and definitive tool for corroborating the initial prognosis and thus potentially remove the need for more invasive procedures, and expedite the commencement of a suitable treatment regimen. It may also avoid the need for a lengthy hospital stay.
  • an expedited conclusive diagnosis may result in the disease being detected at an earlier stage, which may in turn improve overall chances of survival, and/or reduce the risk of hospitalisation.
  • the present invention provides a method for monitoring a cardiovascular disease and/or assessing the severity of a cardiovascular disease in a patient undergoing treatment with an aldosterone antagonist, wherein said method comprises:
  • step (ii) detecting and determining the amount of binding between each monoclonal antibody used in step (i) and peptides in the sample or samples, and (iii) correlating said amount of binding of each monoclonal antibody as determined in step (ii) with values associated with normal healthy subjects and/or values associated with known disease severity and/or values obtained from said patient at a previous time point and/or a predetermined cut-off value.
  • Aldosterone antagonists also known as antimineralocorticoids
  • the aldosterone antagonist is Spironolactone.
  • Preferred embodiments of the second aspect of the present invention are as described above in relation to the first aspect.
  • the present invention provides a method for identifying a patient, with a cardiovascular disease, who is more likely to respond favourably to treatment with an aldosterone antagonist, wherein said method comprises:
  • step (ii) detecting and determining the amount of binding between the monoclonal antibody used in step (i) and peptides in the sample or samples, and
  • step (iii) correlating said amount of binding of the monoclonal antibody as determined in step (ii) with values associated with patients likely to respond favourably to treatment with an aldosterone antagonist and/or with values associated with patients unlikely to respond favourably to treatment with an aldosterone antagonist and/or with a predetermined cut-off value.
  • the aldosterone antagonist is Spironolactone.
  • the immunoassay may be, but is not limited to, a competition assay or a sandwich assay.
  • the immunoassay may, for example, be a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA).
  • the cardiovascular disease may in certain embodiments be heart failure.
  • the cardiovascular disease may be heart failure with a preserved ejection fraction (HFpEF).
  • the patient biofluid sample may be, but is not limited to, blood, serum, plasma, urine or amniotic fluid.
  • the biofluid is serum or plasma.
  • the monoclonal antibody does not recognize or specifically bind to an elongated version of said N-terminus amino acid sequence which is EILGHVPGMLL (SEQ ID No: 28), or to a truncated version of said N-terminus amino acid sequence which is LGHVPGMLL (SEQ ID No: 29).
  • the ratio of the affinity of said antibody for the N-terminus amino acid sequence ILGHVPGMLL (SEQ ID No: 27) to the affinity of said antibody for the elongated N-terminus amino acid sequence EILGHVPGMLL (SEQ ID No: 28), and/or for the truncated N-terminus amino acid sequence LGHVPGMLL (SEQ ID No: 29), is at least 10 to 1 , and more preferably is at least 50 to 1 , at least 100 to 1 , at least 500 to 1 , at least 1 ,000 to 1 , at least 10,000 to 1 , at least 100,000 to 1 , or at least 1 ,000,000 to 1.
  • N-terminus refers to a N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof.
  • Figure 1A Hazard ratio for the primary endpoint per standard-deviation change in fibrosis biomarkers in unadjusted analyses (one model per biomarker).
  • Figure 1 B Hazard ratio for the composite endpoint of death or heart failure admission per standard-deviation change in fibrosis biomarkers in unadjusted analyses (one model per biomarker).
  • Figure 2 Kaplan-Meier survival curves for the primary endpoint among subjects stratified by tertiles of Pro-C6 (left) and Pro-C3 (right).
  • Figure 3 Kaplan-Meier survival curves for the composite endpoint of death or heart failure admission among subjects stratified by tertiles of Pro-C6 (left) and Pro-C3 (right). Examples
  • a monoclonal antibody specific for Pro-C6 was developed as described in WO 2016/156526 (Nordic Bioscience, incorporated herein by reference) using the last 10 amino acids of the type VI collagen a3 chain (i.e. the C-terminus sequence 3168’ KPGVISVMGT ’3177 (SEQ ID No: 1)) as an immunogenic peptide. Briefly, 4-6-week-old Balb/C mice were immunized subcutaneously with 200mI emulsified antigen with 60pg of the immunogenic peptide. Consecutive immunizations were performed at 2-week intervals in Freund's incomplete adjuvant, until stable sera titer levels were reached, and the mice were bled from the 2nd immunization on.
  • the serum titer was detected and the mouse with highest antiserum titer and the best native reactivity was selected for fusion.
  • the selected mouse was rested for 1 month followed by intravenous boosting with 50pg of immunogenic peptide in 100mI 0.9% sodium chloride solution 3 days before isolation of the spleen for cell fusion.
  • Mouse spleen cells were fused with SP2/0 myeloma fusion partner cells.
  • the fusion cells were raised in 96-well plates and incubated in the C02-incubator. Here standard limited dilution was used to promote monoclonal growth.
  • Cell lines specific to the selection peptide and without cross-reactivity to either elongated peptide (KPGVISVMGTA (SEQ ID No: 2), Chinese Peptide Company, China) or truncated peptide (KPGVISVMG (SEQ ID No: 3), American Peptide Company, USA) were selected and sub-cloned. At last the antibodies were purified using an IgG column.
  • the antibodies generated were sequenced and the CDRs determined.
  • CDR-H2 AINPHNGATSYNQKFSG (SEQ ID No: 8)
  • CDR-L1 RSSQRIVHSNGITFLE (SEQ ID No: 4)
  • a monoclonal antibody specific for Pro-C3 was developed as described in WO 2014/170312 (Nordic Bioscience, incorporated herein by reference)using sequence 145’-CPTGPQNYSP-’153 (SEQ ID No: 14) of the a1 chain PIIINP as an immunogenic peptide. Briefly, generation of monoclonal antibodies was initiated by subcutaneous immunization of 4-5 week old Balb/C mice with 200 pi emulsified antigen and 50 pg PIIINP neo-epitope C-terminus sequence (OVA-CGG- CPTGPQNYSP (SEQ ID No: 32)) using Freund’s incomplete adjuvant. The immunizations were repeated every 2 weeks until stable serum titer levels were reached.
  • the spleen cells were fused with SP2/0 myeloma cells to produce hybridoma, and cloned in culture dishes using the semi-medium method.
  • the supernatants were screened for reactivity against calibrator peptide and native material in an indirect ELISA using streptavidin-coated plates.
  • Biotin-CGG- CPTGPQNYSP SEQ ID No: 33
  • the free peptide CPTGPQNYSP SEQ ID No: 14
  • Native reactivity and affinity of the antibody was assessed using different biological materials such as urine, serum, and amniotic fluid (AF) from both humans and rats in a preliminary ELISA using 2 ng/ml biotinylated peptide on streptavidin-coated microtiter plates and the supernatants from growing monoclonal hybridoma cells.
  • Antibody specificity was tested in a preliminary assay using deselection and elongated peptides (i.e. calibrator peptide with ten amino acid substitutions and calibrator peptide with one additional amino acid at the cleavage site, respectively).
  • the isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA). The subtype was determined to be an lgG2 subtype.
  • the antibodies generated were sequenced and the CDRs determined.
  • CDR-L3 FQGAHDPPA (SEQ ID No: 19)
  • a monoclonal antibody specific for C4M was developed as previously described in Sand et. al. 38 (incorporated herein by reference) using the N-terminal neo-epitope sequence 162’- ILGHVPGMLL-’171 (SEQ ID No: 27) generated by MMP-12 cleavage between amino acids 161 and 162 of the a1 chain of type IV collagen as an immunogenic peptide.
  • generation of monoclonal antibodies was initiated by immunization of four to six-week-old Balb/C mice subcutaneously with 200 pi emulsified antigen and 50 pg of the immunogenic peptide (ILGHVPGMLL-GGC-KLH (SEQ ID No: 36)) using Freund’s incomplete adjuvant.
  • Immunizations were performed every 2nd week until stable sera titer levels were reached.
  • the mouse with highest serum titer was selected for fusion.
  • the mouse was rested for one month and then boosted intravenously with 50 m9 of immunogenic peptide in 100 mI 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion.
  • Mouse spleen cells were fused with SP2/0 myeloma fusion partner cells.
  • the resulting hybridoma cells were cloned using a semi-solid medium method, transferred into 96-well microtiter plates for further growth and incubated in a C02 incubator. Standard limited dilution was used to promote monoclonal growth.
  • Native reactivity and peptide affinity of the monoclonal antibodies were evaluated by displacement of native samples (human, rat, and mouse serum, plasma, and urine) in a preliminary indirect ELISA using a biotinylated peptide (ILGHVPGMLL-K-biotin (SEQ ID No: 37)) on streptavid in-coated microtiter plates and the supernatant from the growing monoclonal hybridoma.
  • ILGHVPGMLL biotinylated peptide
  • streptavid in-coated microtiter plates
  • EILGHVPGMLL EILGHVPGMLL
  • the monoclonal antibody was purified from collected supernatant of the selected clones using HiTrap protein G columns and subsequently labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit, according to the manufacturer’s instructions.
  • HRP horseradish peroxidase
  • the monoclonal antibody with the best native reactivity, peptide affinity, and stability was chosen from the antibody-producing clones generated after fusion between mouse spleen cells and myeloma cells.
  • the clones selected were of the lgG1 subtype and the antibodies showed reactivity to healthy human, rat, and mouse serum, as well as human plasma EDTA, and showed no reactivity to the elongated peptide or nonsense peptide.
  • PRO-C3 was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic Bioscience, as described in W02014/170312, and as also detailed in other publications 19 . Briefly, these procedures were as follows:
  • TMB tetramethylbenzinidine
  • PRO-C6 was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic Bioscience, as described in WO2016/156526, and as also detailed in other publications 39 . Briefly, these procedures were as follows:
  • ELISA-plates used for the assay development were Streptavidin-coated from Roche (cat.: 1 1940279). All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, (CA, USA). We labeled the selected monoclonal antibody with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (Innovabioscience, Babraham, Cambridge, UK).
  • HRP horseradish peroxidase
  • a 96-well streptavidin plate was coated with biotinylated synthetic peptide biotin-KPGVISVMGT (SEQ ID No: 38) (Chinese Peptide Company, China) dissolved in coating buffer (40 mM Na 2 HP0 4 , 7 mM KH 2 P0 4 , 137 mM NaCI, 2.7 mM KCI, 0.1 % Tween 20, 1 % BSA, pH 7.4) and incubated 30 minutes at 20°C.
  • coating buffer 40 mM Na 2 HP0 4 , 7 mM KH 2 P0 4 , 137 mM NaCI, 2.7 mM KCI, 0.1 % Tween 20, 1 % BSA, pH 7.4
  • C4M was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic Bioscience, as described in Sand et al 38 . Briefly, these procedures were as follows:
  • a 96-well streptavidin-coated microtiter plate (cat. no. 1 1940279, Roche Diagnostics, Hvidovre, Denmark) was coated with 100 pi biotinylated peptide (ILGHVPGMLL-K-biotin (SEQ ID No: 37)) dissolved in coating buffer (50 mM Tris, containing 1 % bovine serum albumin, 0.1 % Tween-20, and 0.4% bronidox (BTB), pH 8.0) and incubated for 30 minutes at 20°C.
  • coating buffer 50 mM Tris, containing 1 % bovine serum albumin, 0.1 % Tween-20, and 0.4% bronidox (BTB), pH 8.0
  • results were analysed spectrophotometrically at 450 nm with 650 nm as the reference using an ELISA microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).
  • a standard curve was performed by serial dilution of the standard peptide and plotted using a 4-parametric mathematical fit model.
  • TOPCAT was a multi-center, international, randomized, double-blind, placebo-controlled trial of spironolactone that enrolled 3445 adults with HFpEF across >270 clinical sites in 6 countries from August 2006 until January 2012.
  • the primary results of the trial have been previously published 42 . All study participants provided written informed consent.
  • Inclusion criteria for TOPCAT were as follows: age >50 years; diagnosis of HF based on at least 1 HF symptom at the time of study screening and at least 1 HF sign within the 12 months before screening; left ventricular EF >45% (per local reading); at least 1 HF hospitalization in the 12 months before study screening or BNP (B-type natriuretic peptide) >100 pg/mL or NT-proBNP (N-terminal pro-BNP) >360 pg/mL (in the absence of an alternative explanation for elevated natriuretic peptide level) within the 60 days before screening; and serum potassium ⁇ 5.0 mmol/L before randomization 40 ' 42 .
  • Exclusion criteria have been published in detail previously 40 but included severe systemic illness with a life expectancy of ⁇ 3 years, significant chronic pulmonary disease, infiltrative or hypertrophic cardiomyopathy, constrictive pericarditis, previous cardiac transplant or LV assist device, known chronic hepatic disease, severe chronic kidney disease (defined as estimated glomerular filtration rate [eGFR] ⁇ 30 mL/min per 1 .73 m 2 or serum creatinine >2.5 mg/dL), a history of significant hyperkalemia, known intolerance to aldosterone antagonists, and recent myocardial infarction, coronary artery bypass grafting, or percutaneous coronary intervention.
  • eGFR estimated glomerular filtration rate
  • the primary goal of the trial was to determine if spironolactone was associated with a reduction in the composite outcome of cardiovascular mortality, aborted cardiac arrest, or heart failure hospitalization. All HF hospitalizations were adjudicated by a clinical end point committee at Brigham and Women’s Hospital, blinded to study-drug assignments, according to prespecified criteria, as previously described 40 . In this analysis, we examined the relationship between biomarkers and tissue fibrosis and: (1) The primary endpoint, as defined above; (2) A composite endpoint of death or heart failure hospitalization, which is increasingly utilized in HFpEF studies 43 .
  • Pro-C3, Pro-C4 and Pro-C6 Specific biomarkers of collagen formation (Pro-C3, Pro-C4 and Pro-C6) and degradation (C3M, C4M and C6M) were measured using enzyme-linked immunosorbent assays (ELISA).
  • ELISA enzyme-linked immunosorbent assays
  • the Pro- C3, Pro-C6 and C4M ELISAs were carried out as described supra (see Examples 4, 5 and 6, respectively).
  • Pro-C4 is a known biomarker of collagen type IV formation, and the Pro-C4 ELISA was carried out in the manner described in Leeming et al 44 .
  • C3M and C6M are known biomarkers of collagen type III degradation and collagen type VI degradation, respectively, and the C3M and C6M ELISAs were carried out in the manner described in Barascuk et al 45 and Juhl et al 46 , respectively.
  • NT-proBNP levels were measured using a validated Luminex ® Bead-Based multiplexed assay (Bristol Myers-Squibb; Ewing Township, NJ).
  • Participant characteristics were summarized using mean (SD) for normally distributed variables and median (interquartile range) for non-normally distributed continuous variables. Categorical variables are expressed as counts (percentages). Subjects enrolled in the Americas who had available samples for measurement of the biomarkers of interest vs. those who did not were compared. The non-paired t test for normally-distributed variables, the Kruskal-Wallis test for non-normally distributed variables and the chi-square test or Fisher’s exact test, as appropriate, for categorical variables were used.
  • biomarkers The relationship between biomarkers and the primary outcome (cardiovascular death, aborted cardiac arrest, or heart failure hospitalization), as well as the composite of HF hospitalization or all-cause death, were assessed using Cox regression. Kaplan-Meier survival curves for tertiles of each biomarker were constructed, and these were compared using the log-rank test.
  • Adjusted Cox models were built, as appropriate, to assess whether unadjusted associations are independent of confounders, including: (1) the MAGGIC risk score, which incorporates multiple demographic, clinical and laboratory variables (Model 1) 47 ; (2) The MAGGIC risk score plus NT- proBNP levels (Model 2); (3) Important individual clinical covariates chosen a priori, including age, sex, diabetes mellitus status, estimated glomerular filtration rate, systolic blood pressure (SBP), and NYHA class lll/IV and history of myocardial infarction (Model 3). Hazard ratios for all biomarkers are standardized (expressed per standard-deviation increase, or 1 -point increased in the z score) in order to provide an intuitive comparison between the biomarkers.
  • NYHA class lll-IV 540 (34.70%) 80 (38.83%) 0.2434 Myocardial Infarction 313 (20.09%) 46 (22.33%) 0.4529 Stroke 143 (9.18%) 15 (7.28%) 0.3703 COPD 269 (17.27%) 22 (10.68%) 0.0167
  • Beta Blockers 1215 (77.98%) 172 (83.50%) 0.0698
  • Glucose-lowering agents 628 40.31 %) 91 (44.17%) 0.2885 ACE Inhibitors or ARBs 1240 (79.59%) 154 (74.76%) 0.1094
  • Figure 1 A shows standardized hazard ratios for all examined fibrosis biomarkers for the primary endpoint in unadjusted analyses (one model per biomarker).
  • Figure 1 B shows corresponding standardized hazard ratios for death or heart failure admission.
  • Figure 2 shows Kaplan-Meier survival curves for the primary endpoint corresponding to the tertiles of Pro-C6 (left) and Pro-C3 (right), respectively.
  • Pro-C6 stratified subjects across a broad range of absolute risk. There was graded pronounced reduction in event-free survival from the lowest tertile (Pro-C6 ⁇ 1 1.0 ng/ml) to the highest tertile (Pro-C6 > 16.0 ng/ml) of Pro-C6. For Pro-C3, only the highest tertile (Pro-C3 > 14.0 ng/ml) demonstrated a pronounced reduced event-free survival. A similar pattern was found for death or heart failure admission, as shown in Figure 3.
  • pro-C6 as a continuous variable
  • pro-C3 level >14 ng/ml_ (highest tertile of distribution, expressed as a binary variable)
  • pro-C6 but not pro-C3 status was independently predictive of the primary endpoint and of death/HF admission.
  • the Harrel’s C-statistic was much greater for a model including only pro-C6 alone (0.705) than for models including the MAGGIC risk score (0.552), the MAGGIC risk score plus BNP (0.582), or a combination of clinical variables included in adjusted model 3 (0.64).
  • the Harrel’s C-statistic was much greater for a model including only pro-C6 alone (0.707) than for models including the MAGGIC risk score (Adjusted model 1 : 0.
  • Adjusted Model 1 adjusted for the MAGGIC risk score.
  • Adjusted Model 2 adjusted for the MAGGIC risk score and NT-proBNP levels.
  • Adjusted Model 3 adjusted for age, sex, diabetes mellitus, estimated glomerular filtration rate, systolic blood pressure (SBP), NYHA class lll/IV and history of myocardial infarction.
  • biomarkers of ECM turnover were studied. It was demonstrated that pro-C6 and pro- C3, biomarkers of fibrogenesis assessed by type VI and III collagen formation, respectively, predicted the risk of incident cardiovascular events, as well as a composite of all-cause death/HF-related hospitalization in this population. Pro-C6, in particular, was a strong independent predictor of these outcomes and stratified subjects across a broad range of absolute risk. Pro-C6 alone performed better as a predictor of outcomes than the MAGGIC risk score, NT-ProBNP or a combination of clinical variables.
  • pro-C6 appears to be a particularly strong and robust independent predictor of outcomes in HFpEF. Pro-C6 may thus be useful in the diagnosis of HFpEF, for the identification of good candidates for antifibrotic therapies, and/or for monitoring and characterizing the efficacy of such therapies.
  • a particularly interesting finding of the present study is the highly significant interaction between C4M and the risk modification associated with randomized treatment with spironolactone.
  • biomarkers of collagen turnover can identify individuals who benefit from spironolactone.
  • the present study is the first that reports an interaction between biomarkers of collagen turnover and the reduction in the risk of clinical events associated with spironolactone therapy. It was found that lower C4M levels were associated with a greater reduction in risk associated with spironolactone randomization. C4M is a marker of collagen degradation; therefore, lower levels indicate reduced degradation and thus increased collagen accumulation, which is a therapeutic target of spironolactone.
  • fibrogenesis assessed by Pro-C6 is strongly and independently predictive of a poor prognosis in HFpEF.
  • low levels of C4M appear to identify patients with HFpEF who exhibit particularly favorable responses to aldosterone antagonists (mineralocorticoid-receptor antagonists).
  • Lam CS Donal E, Kraigher-Krainer E, Vasan RS. Epidemiology and clinical course of heart failure with preserved ejection fraction. Eur J Heart Fail 201 1 ; 13: 18-28.

Abstract

L'invention concerne une méthode de d'immunoessai permettant de détecter et/ou de surveiller une maladie cardiovasculaire chez un patient et/ou d'évaluer la probabilité ou la gravité d'une maladie cardiovasculaire chez un patient, comprenant la mise en contact d'un échantillon de fluide biologique provenant d'un patient avec un anticorps monoclonal qui se lie spécifiquement à un épitope C-terminal du domaine C5 de la chaîne a3 du collagène de type VI, et/ou la mise en contact d'un échantillon de fluide biologique provenant du patient avec un anticorps monoclonal qui se lie spécifiquement à un néo-épitope C-terminal du propeptide N-terminal du collagène de type III.
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